Unit 2 - Cell Cycle Flashcards
key tasks for proliferating cells
- replicate entire genome (ONCE) - fertilised egg → entire organism, cellular regeneration, in response to injury
- separate duplicated chromosomes equally into daughter cells (UNLIKE stem and germ cells)
- co-ordinate cell growth and proliferation by ensuring cells have enough energy and metabolites before entering into the processes
key events of the cell cycle
Gap phase G1
cell growth and regulatory events (checkpoints)
synthesis phase (S)
DNA replication occurs
gap phase 2 G2
cell growth and regulatory events (checkpoints)
M phase - mitosis
chromosomes are segregated and cells divide
when may cells exit cycle into G0
if terminally differentiated, senescent or if inhibitory signals are received
cell cycle timing
varies between cells depending on function and origin
Our genetic material is very large and will take a while to duplicate accurately, and segregate it between daughter cells
enzymes are conserved
DNA polymerases
highly conserved enzymes
require a 3’-OH group for activity and so require a primer
needed to extend DNA strands in a 5’ → 3’ direction
replicative polymerases = Pol δ and Pol ε
DNA replication overview
DNA is unwound and RNA primer molecules bound are synthesised by primase
primers are then extended by replicative polymerases (δ or ε) in a 5’ → 3’ direction
⇒ lagging strand is discontinuously synthesised
also leads to ‘end replication problem’ which is solved by telomerase
primase function
extend chains of DNA against the template provided by the pre-existing chromosome
Okazaki fragments
discontinuous segments being synthesised from RNA primer
telomerase function
allow intact replication of RNA ends
arises from the need of our polymerases to have our primer structure
steps in DNA synthesis - lagging strand
- helicase unwinds DNA, RPA loads - Pol α-primase synthesises a short primer
- Pol α displaced and Pol δ/Pol ε loaded
- Pol δ/Pol ε extend the primer
- downstream primer is removed (nuclease)
- Okazaki fragments are ligated
SIMILAR PROCESS OCCURS ON LEADING STRAND, ensuring both strands of DNA molecule are going to be duplicated completely
Pol α-primase function
- Synthesise a short RNA primer against the template provided by the original DNA strand
- Allow replicate of polymerases (Pol delta or epsilon) to extend in 5’ to 3’ direction from the 3’-OH in a process that will give rise to the new DNA strand
- Primer is then removed by nuclease activty
- Individual extended primers, now the actual DNA sequences, are ligated together again to give a continuous new DNA strand that will base pair with the original DNA strand and will be complementary to it due to extend of polymerase
M phase overview
chromosomes condense and attach to microtubules from mitotic spindle
all chromosomes must be attached before sister chromatids can separate
each daughter cell receives 1 set of chromosomes
chromosome segregation is irreversible so this process is highly-regulated
what happens when all chromatids have attached to the poles of mitotic spindle
the sister chromatids - duplicated pairs of chromosomes - will separate from one another
(held together after replication but prior to separation during mitosis)
6 phases of mitosis
prophase
prometaphase
metaphase
anaphase
telophase
cytokinesis
prophase
chromosomes begin to condense
requires condensin and DNA topoisomerase II
the (duplicated) centrosomes separate
histones undergo mitosis-specific modifications
Lose their diffused localised volume and begin to adopt a more tightly packed conformation that is mechanically necessary for them to migrate to opposite poles later in mitosis
intact nuclear envelope
what makes up chromatin
histones
prometaphase
microtubules from opposite spindle poles (centrosomes) bind chromosomes at kinetochores (waist) to initiate bipolar orientation
nuclear envelope breakdown occurs - can now spread out into the cell
metaphase
all chromosomes have made bipolar attachemnts to spindle poles
chromosomes align at metaphase plate
what is the key tightly-regulated step in mitosis
between metaphase and anaphase
anaphase
chromatids separate and move toward the opposite spindle poles - poles separate
nuclear envelope reassembly commences - reassembles around the chromosomes as they move apart
telophase
nuclear envelope reassembles around sister chromatids
poleward movement of chromosomes continues
cleavage plane is specified (Plane along which the cytoplasm will eventually separate)
cytokinesis
separation of daughter cells
formation of the cleavage furrow by a contractile ring of actin filaments (between the 2 masses of chromosomes)
chromosomes decondense and nuclear structures reform
vertebrate centromeres
the primary constriction in higher eukaryote chromosomes
heterochromatin region
centromeric heterochromatin carries the kinetochore
megabase arrays of highly repetitive satellite DNA
chromosomes contain varying amounts of satellite DNA of varying sequence
principal component of human CEN sequences is α-satellite DNA - monomer structure = 171 bp - arranged into complex repeats (2-32 monomers/repeat)
principal component of human CEN sequence
α-satellite DNA
key function of centromeric DNA sequence
it carries proteinic structure known as kinetochore
microtubules attach to kinetochore
vertebrate kinetochore
complex proteinaceous structure that controls chromosome-microtubule attachment and mitotic spindle assembly
trilaminar structure
kinetochore assembly on centromeric chromatin = temporally-regulated process involving several pathways
controls attachment of kinetochore to the microtubules
inner plate connects to chromosomes
key regulatory steps in cell cycle
making sure DNA replicates completely and once only - S phase control
ensuring DNA is intact before mitosis begins - G2 phase delays
making sure all chromosomes are segregated equally to 2 daughter cells - spindle checkpoint
overall co-ordination/timing
S phase control
pre-replicative complex binds to origins of replication (licensing) during late M/G1
licensing origins of replication are bound by initiator proteins (a large protein complex)
once activated, pre-replicative complex disassembles and cannot reassemble to reactivate an origin until the next cell cycle
each of the origins can fire only once per cell cycle
Orc1-6
Controlling the activation processes at the origins of replication
Cdc6 and Cdt1
Binds to origins of replication through out the chromosomes of the cell during late M phase/beginning of G1
Ensures the processes of replication can occur at each of the origins
Monitoring/regulating DNA integrity
centrosome
2 centrioles (barrels of microtubules composed of tubulin and centrin)
surrounded by cloud of pericentriolar material (PCM)
PCM contains γ-tubulin ring complex that nucleates microtubules
contents of PCM
γ-tubulin ring complex that nucleates microtubules
what do microtubules eventually connect to
kinetochore - forms on centromeric DNA
what are centrioles
microtubule structures
contain tubulin in polymer and Ca2+ binding protein centrin
centrosome duplication cycle
Ensures divisions organised by central poles are bipolar - pulling chromosomes in 2 directions
mitotic spindle assembly
unstable centrosome microtubules are captured and stabilised by kinetochore binding until all are assembled
interpolar microtubule motors separate the spindle poles
bipolar orientation of chromosomes allows equatorial positioning
spindle assembly/metaphase checkpoint
- translation of absence of appropriate spindle-kinetochore interactions into a biochemical signal (wait anaphase) - If they lack microtubule attachments signal will block cell entry into anaphase
- satisfaction of this checkpoint requires both occupancy of kinetochores by microtubules and inter-kinetochore tension
- Sister kinetochores are attached to opposite poles of mitotic spindle - alleviates ‘wait anaphase’ signal and allow cell to progress
- defective checkpoint signalling implicated in tumorigenesis
control of cell cycle - what are the key regulators
how are they regulated
cyclin dependent kinases (CDKs)
cyclin levels oscillate during cell cycle
different cyclins are active at different stages in the cell cycle, being regulated through transcription and through degradation
key transcription regulators determine directionality of cell cycle e.g. pRb which controls E2F family transcription factors in G1/S, thus regulating cyclins A and E
CDK-cyclin activities are also regulated through phosphorylation
oscillations of cyclins through a cell cycle
CDK activities are controlled by [regulated] cyclin levels
key transcriptional regulators determine cell cycle direction e.g. pRb, which controls E2F family transcription factors in G1/S, thus regulating cyclins A/E
(purple controlled through degradation - anaphase promoter complex)
pRb
controls E2F family transcription factors in G1/S, thus regulating cyclins A/E
cyclin/CDK combinations and activities
Cdk1-cyclin B phosphorylation targets grouped on the basis of
protein function
Cdk2-cyclin A phosphorylation targets grouped on the basis of protein function
activation of CDK cyclin pair determines the cell cycle stage
key target sites in regulated activation of Cdk1
Active site cleft can be controlled by certain enzymes that can be phosphorylated in activating/repressing state
feedback loops that control activation of Cdk1
targeting of the cell cycle in cancer therapy
anitmitotics
vincristine/vinblastine and taxol
impact proliferative status of cells - microtubules
alkylating agents/Pt drugs
DNA damage
radiotherapy - Topo II inhibition (doxorubicin)
stop cells from entering into mitosis - perhaps cell death directly
antimetabolites
block nucleic acid synthesis
impedes processes for successful S phase
challenge of converting understanding of cell cycle into therapeutics
although CDKs are attractive targets, pan-CDK inhibitors are not established for use in the clinic, despite numerous trials
problems:
non-specificity of action (similarity of kinase catalytic sites), redundancy and unexpected side effects (involvement in other activities)
targeting other components of the cell cycle machinery has been more successful so far e.g. anti-replication drugs e.g. 5-FU/gemcitabine
example of anti-replication drug
5-FU
gemcitabine
3 mechanisms of DNA repair
base excision
nucleotide
mismatch
activating point mutations in RAS - consequences
all compromise the GTPase activity of RAS
this prevents the hydrolysis of GTP on RAS, causing RAS to accumulate in the GTP-bound, active form
almost all RAS activation in tumours is accounted for by mutations in codons 12, 13 and 61
Ras mutations in cancer
Mutations in a very small number of codons in human genome causes this
Only 3 bps
codons involved in RAS activation
mutations in codons 12, 13 and 61
causative DNA damage and cancer
major contributory factor
UV damage and skin cancer
cigarette smoke and lung cancer
therapeutic DNA damage and cancer
major anti-cancer strategies kill tumour cells by DNA damage
chemotherapy e.g. cisplatin
radiotherapy
sources of DNA damage
endogenous sources
free radicals
spontaneous base deamination/depurination
DNA replication errors (normal metabolism)
exogenous sources
radiation (ionising, UV)
chemicals (benzo[a]pyrene in cigarette smoke; aflatoxin; anti-cancer drugs)
multiple pathways of DNA repair
different lesions are repaired through different repair mechanisms
double-strand break results in
discontinuities in the sugar phosphate backbone of the double helix
base excision repair
DNA base damage occurs continuously in our cells
generation of abasic (apurinic/apyrimidinic ⇒ AP) sites through hydrolytic cleavage of N-glycosidic bond - 2,000-10,000/cell/day
cytosine → uracil
100-500/cell/day
adenine → hypoxanthine
10-50/cell/day
oxidative damage example
8-oxo-deoxyguanine (8-oxo-dG)
100-500/cell/day
alkylation damage example
O6-methylguanine
what do deamination changes cause
inappropriate base pairing, which is mutagenic
mechanism of BER
DNA glycosylase recognises damaged base and removes it, leaving abasic (AP) site
AP endonuclease cleaves the DNA at this AP site
DNA polymerase carries out repair synthesis
DNA ligase rejoins sugar-phosphate backbone
nucleotide excision repair
removes DNA lesions that strongly distort DNA structure
especially UV-induced lesions
beach in strong sunlight - 40,000 damaged sites per hour in exposed epidermal (skin) cell
due to UV light (200-320 nm)
most UV-C light (100-290 nm) is absorbed by ozone layer and air will absorb UV to 200 nm
xeroderma pigmentosum
lack of DNA repair of UV-damafe results in skin cancer susceptibility
XP is a rare human disease caused by inherited mutations in XP genes
leads to extreme susceptibility to skin cnacer (melanoma, squamous cell carcinoma) arising from solar UV-induced DNA damage
NER mechanism
DNA distortion from the damaged bases is recognised by a complex containing XPA and XPC (recognise the damaged DNA)
in transcribed DNA, the stalled RNA polymerase can act as a recognition signal
DNA helix is unwound by XPB/XPD
XPF/Ercc I and XPG nick the DNA 5’ and 3’ of the lesion
DNA polymerase δ or ε synthesises the excised sequence and DNA ligase seals the nicks
mismatch repair
human genome - 3 x 109 bps
genome must be accurately replicated (during S phase) each time a cell divides
occasionally DNA polymerase (particularly pol δ) makes an error in copying DNA
mismatch repair required to correct these errors
mechanism of MMR
mismatch recognition proteins (MSH2/MSH6) detect the error
a sliding clamp is formed to find a single-strand nick (newly synthesised DNA)
DNA is exonucleolytically degraded until the mismatch
repair synthesis is performed by DNA polymerase δ or ε and DNA ligase seals the nicks
MMR and cancer
defective MMR causes microsatellite instability
hereditary non-polyposis colon cancer results from germline mutations in MSH2 and MLH1
e.g. 9/11 HNPCC cell lines studied had a mutation in a stretch of the type II TGF-β receptor sequence
this causes the instability of the (truncated) receptor, making cells insensitive to the growth inhibition signals of TGF-β
DNA double-strand breaks
discontinuities in both strands of the DNA double helix
particularly hazardous to cells because of the risk of translocations of the loss of genetic material
TELOMERE = a special protective nucleoprotein complex at the ends of linear chromosomes to ensure that they are not treated as broken ends
telomere
a special protective nucleoprotein complex at the ends of linear chromosomes to ensure that they are not treated as broken ends
sources of DSBs
ionising radiation (X rays, gamma rays) - short wavelength, high energy
generates free radicals in cells
leads to DNA damage - single-strand breaks (ssbs) and double-strand breaks (dsbs) in DNA
carcinogenic
leads to cell death ⇒ used in radiotherapy
localised doses can be very high (50Gy), but > 5Gy whole-body irradiation is lethal
radiomimetic chemotherapeutic drugs (topoisomerase II inhibitors e.g. doxorubicin/adriamycin)
repair of single-strand breaks - rapid in cells - requires poly (ADP-ribose) polymerase (PARP)
examples of topoisomerase II inhibitors
doxorubicin
adriamycin
non-homologous end-joining
repairs DSB with no requirement for major homology - potentially mutagenic
principle DSB repair mechanism in mammalian cells
breaks are bound by Ku70/Ku80 dimer to initiate repair
broken ends are religated by DNA ligase IV/XRCC4
this process is critical in V(D)J recombination during immune system development
what is lack of sequence homology useful in
allowing us to have a large immune repertoire
homologous recombination
accurate, template-directed repair mechanism - with use of intact sister chromatid available - Limited to post-replicational phase - needs intact alternative template for repair
initiated by resection at break to expose a tract of ssDNA
Rad5 I recombinase forms a nucleoprotein filament on ssDNA which carries out a homology search
strand invasion allows templated DNA synthesis and the resulting Holliday junction is cleaved to yield repaired sequence
BRCA2 is a key component in HR
DNA damage responses and disease
germline DNA repair defects in disease (cancer predisposition and other non-cancer diseases)
spontaneous mutations arising in carcinogenesis
exploitation of DNA repair pathways to kill tumour cells
germline DNA repair gene defects predisposing to cancer
DNA repair defects differ among cancer types
exploiting our understanding of DNA repair
synthetic lethality
spontaneous breaks that occur during normal DNA replication are repaired by HR and Parp-I dependent ssb repair (Normally available to cell - if spontaneous break occurs EITHER OR)
BRCA2-defective cells have defective HR so rely on Parp I to repair such breaks
inhibition of Parp I in BRCA2-negative cells is a specific and potent killing mechanism
BRCA2 cells do not have homologous recombination
Rely completely on PARP I
Inhibition of this pathway makes the spontaneous lesions that occur during normal replication lethal to the cells
